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33 4

0026-895X!96/020334-08$3.OO/O

C opyr igh t © by The Am e ric an S ocie ty fo r Pha rm aco logy an d Expe rim en tal T h erapeu tics

A ll righ ts of rep ro duc tion in any fo rm rese rved .

M O LECU LAR PH ARM A CO LO GY , 50 :33 4 -341 (1996 ).

A ttenua tion o f Induc ib le N itr ic O x ide Syn thase G ene

Exp ress ion b y L \9 -Te trahyd rocannab ino l Is M ed ia ted th rough

the In h ib ition o f N uc lear Fac to r-KB /R e l A c tiva tion

YOUNG J . JEON , K YU -H . YA NG , J IM T . PU LA SK I, a nd NORBER T E . K AM IN SK I

Depa rtm ents o f P ha rm aco logy and Tox ico logy and P atho logy, M ich igan S ta te Un ivers ity , East Lans ing , M ich igan (J .T .P ., N .E .K .), and

D epa rtm ent o f B io log ica l S c iences, Ko rea Advanced Ins titu te o f Sc ience and Techno logy, Tae jon, K orea (Y .J .J ., K -H . Y .)

Rece ive d M arch 5 , 19 96 ; A ccep te d M ay 2 , 19 96

SUMMARY

9 -Te trahyd rocannab ino l (9 -THC ), a p ro to typ ic c ompound

be lon g ing to the fam ily of agen ts k now n as cannab ino id s,

pro duces a w id e va rie ty o f b io log ica l effec ts , in clu d ing inh ib i-

tion o f imm une func tion . The pu ta tive m echan ism fo r cannab i-

no id b io log ical ac tion invo lves b ind ing to cann ab ino id recep to r

types 1 an d 2 (C B1 and CB 2) to nega tive ly regu la te adeny la te

cyc lase and inh ib it in trace llu la r s igna ling v ia the cA M P cas-

cad e. In the cu rren t stud y , w e sh ow tha t z 9 -TH C p ro duces a

m arked in h ib ition of in duc ib le n itr ic ox id e sy n thase (iN O S ) tran -

sc rip tion and n itric ox ide p roduc tion b y the m acrop hage line

RAW 264 .7 inresponse to lipop o lysaccharide (LPS) . A na lys is o f

RAW 264 .7 ce ll RN A dem ons tra ted transc rip ts fo r CB 2 bu tot

CB 1 . T rea tm en t o f RAW 264 .7 w ith z 9 -TH C inh ib ited fo rsko lin -

s tim ula ted cA M P produ c tion in a dose-rela ted m anner, v er ify -

ing the ex press ion of fu nc tion a l cannab ino id recep to rs by th is

ce ll line . iN O S tran sc rip tion , w hich is regu la ted in part b y the

nu clea r fac to r-K B /R el (N F -K B /R el) fam ily of transc rip tion

to rs, has been show n to be und er the con tro l o f th e

s igna lin g cascad e. W e d em onstra te th a t 9 -TH C inh ib its

ac tiv a tion and b ind ing of N F -K B /R el p ro te in s to th eir cogn

DNA site , K B , in re spon se to L PS stim u la tion . L PS treatm en t

RA W 264 .7 ce lls a lso induced the ac tiv ation of the cA M P

cade , a s in d icated by an inc rease in b ind in g o f n uc lea r fac

to the cAM P response e lem en t. A c tiv a tion of G R E bi

pro te ins w as inh ib ited by z 9 -THC . Forsko lin treatm en t o fAW

264 .7 ce lls induced bo th KB and cAM P resp onse e lem en t b in d -

ing ac tiv ity and w as lik ew ise inh ib ited by 9 -TH C . C ollec tive ly ,

th is se ries o f exp er im en ts ind ica tes tha t N F-K B /R el is pos itiv

regu la ted by th e cA M P cascad e to he lp in itia te iN O S

exp re ssio n in re sp onse to L PS s tim u la tion of m ac roph ag es.

Th is ac tiva tion of iN OS is a ttenua ted by z 9 -THC th ro ugh

inh ib ition of cA M P signa ling .

Curren tly , tw o m ajo r ty pes o f cannab in o id recep to rs , CB 1

and CB 2, h av e b een iso lated an d clo ned . B o th recep to rs are

invo lved in m ed ia tin g the d iv erse b io lo g ical actio ns o f cann a-

b ino id com pounds, inc lud ing imm une su ppre ssion and alte r-

a tion s in cen tra l ne rvo us sys tem fun c tion . CB1 is expressed

in g rea te st abun dance w ith in the b rain (1 , 2 ) b u t is a lso

p resen t a t low leve ls in p e riph era l tissues, no tab ly in sp leen

(3 ). CB 2 h as b een iden tif ied on ly w ith in the imm une sy stem

(4) . B o th recep to r typ es a re n eg ativ e ly co up ledo adeny la te

cyc lase th rough a pertuss is tox in -sens itiv e G pro te in (5 ) and

inh ib it the cA M P sign aling cascade . S truc tu re -ac tiv ity s tu d-

ies sh ow a stron g co rre la tion am ong the m agn itude of inh i-

b ition b y cannab in o ids on adeny la te cy c la se ac tiv ity , th eir

This w ork was supported by fu nds from N ation al Ins titu te o n D rug A buse

G rants D A07908 and D A 09171 .

b ind in g affin ity , an d the ir re spec tive ran k ord er o f p o tency

modify ing b io log ica l re spo nses (6 ).

A lthough the m echan ism for imm un e suppression b y

nab in o ids has no t been c lea rly estab lished , d is rup tion o

cAM P signa ling cascade in itiated by recep to n /ligand in te rac -

tion is a c ritica l even t in the su ppress ion of a n um ber

imm une responses. T h is is m ade ev id en t by the ab ility

memb ran e -p e rmeab l e cA M P ana lo gs to rev erse cannab ino id -

m edia ted in h ib itio n ofimm une re spon ses, inc lud in g the

sheep en y thnocy tes 1 gM antibod y-fo rm ing ce ll respo nse and

lym phopro life ratio n (6 ). S im ila rly , A D P -n ibosy la tio n of inh ib -

ito ry G pro te ins in sp leno cy tes b y pertussis tox in

can nab in o id inh ib ition of bo th ad en y la te cyc lase ac tiv ity

an tibod y-fo rm in g ce ll respon ses (6 ) . C an nab ino id recep to r

expre ssion h as b een iden tif ied in a ll o f the m a jo r im m u

log ica l ce lls typ es , in clu d ing B and T ce lls , m onocy te s,

ABBREV IA T IO NS : CB 1 , cannab ino id rec ep tor ty p e 1 ; C B2 , cannab ino id re c ep to r ty p e 2 ; 9 -TH C , 9 - te tra h yd ro cannab ino l; PM A , pho r

my ris ta te -1 3 -a ce ta te ; R PM I 1640 , R o sw ell P ark M em o ria l Ins titu te 1 640 ; b p , ba se pa ir(s ); R T -PC R , re ve rse tra nsc rip tio n -p o lym e ra se cha in

rea c tion ; iN O S , in d uc ib le n itr ic o x id e syn tha se ; G RE , cAM P re spon se e lem en t; N F -KB , nu c lea r fac to r fo r im m unog lo bu lincha in in B ce lls ; LPS ,

lip o po lys accha rid e ; NO , n itr ic o x id e ; PK A , cAM P -dependen t p ro te in k ina se ; PD TC , p y rro lid ine d ith io ca rbam a te ; IS , in te rna l s ta nd a rd ;

e lec troph ore tic m obility sh iftassay ; BSA , bov ine se rum a lbum in ; GREB ; cAM P response e lem en t b ind ing p ro te in ; A TF , ac tiva ting transcr ip

fac to r; IF N-y , in te rfe ro n -y ; H EPES , 4 -(2 -hydroxye thy l)-l -p ipe raz inee thanesu lfon ic ac id .

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9 -TH C In h ib it s LP S -ln d u c e d N O S a n d N F -.c B /R e l B in d in g 335

mast cells, providing a rationale for the sensitivity of the

immune system to modulation by cannabinoids (3, 4, 7, 8) .

Furthermore, it is likely that immunocellular responses reg-

ulated by cAM P signaling w ill exhibit sensitivity to cannabi-

noids and account for the w ide range of immunological re-

sponses effected by this class of compounds.

W e investigated the effect of 9-THC on the LPS-induced

NO response in macrophages, an important aspect of inflam-

mation and innate host defense to bacterial pathogens. Stim-

ulation of munine macrophages by LPS results in the expres-

sion of an iN OS, which catalyzes the production of large

amounts of NO from L -anginine and molecular oxygen (9).

N O, in turn, participates in the cytolytic function of macro-

phages (10). T he promoter ofthe munine gene encoding iNOS

contains two KB binding sites, located at 55 and 971 bp

upstream of the TA TA box, respectively (11) . I t has been

reported that protein binding to the K B site is necessary to

confer inducibility by LPS (12). T he NF-K BfRel family of

transcription factors are pleiotropic regulators of many genes

involved in immune and inflammatory responses, including

iN OS (12, 13). N F-K BIRe1 exists in the cytoplasm of unstimu-

lated cells in a quiescent form bound to its inhibitor,KB .

M acrophage activation by certain external stimuli results in

the phosphorylation of 1K B by PICA , thus releasing the active

D NA -binding form ofNF-K BIRe1 to translocate to the nucleus

to bind KB motifs in the regulatory region of a variety of

genes (14) . LPS treatment of macrophages activates both

protein kinase C and PKA , the latter being induced by an

elevation of intracellular cAM P (15, 16). A lso, interleukin-1,

an inflammatory cytokine induced by LPS, contributes to the

elevation of cAM P. The coordinate activation mediated

through LPS and interleukin-1 is followed by increased iNOS

expression and nitrite formation (17, 18). T hese results sug-

gest that (a) the cAM P signaling cascade is involved in both

NF-K BIRe1 activation and iN OS induction after LPS stimu-

lation and (b) the LPS-induced NO response is a likely can-didate for modulation by cannabinoids. H ere, we examine the

role ofcAM P signaling on LPS-induced iNOS activity and NO

formation in the macrophage cell lineAW 264.7 by using

the adenylate cyclase inhibitor # {176} -THC.

Ma t e r ia ls a n d Me t h o d s

R eagents. z#{ 176} -TH Cwas provided by the National Institute on

D rug A buse (N ational Institutes of H ealth, Rockville, M D ). LPS,

PDTC , and 8-bromo-cAM P were purchased from Sigma Chemical

(St. L ouis, M O); forskolin was purchased from CalB iochem (L a Jolla,

CA ); PCR reagents were purchased from Promega (M adison, W I );

and reagents used for cell culture were purchased from GIBCO-BRL

(Grand Island, N Y ).

Animals. V irus- free 4-6-week-old female B6C3F1 mice were pur-

chased from the Frederick Cancer Research Center. On arrival,

randomized mice were transferred to cages containing sawdust bed-

ding (five mice/cage), given food (Purina Certified Lab Chow) and

water ad l i bi tum, and used for experimentation when their body

weight was 17-20 g. Animal holding rooms were maintained at

21-24#{ 176} and 40-60% relative humidity w ith a 12-hr light/dark cycle.

C el l cu l t u r e. T h e per i t on eal m acr ophages andAW 264.7 cells

(No. T IB-71; American T ype Culture Collection, Rockvil le, M D ) were

grown in RPM I 1640 supplemented w ith 10% fetal bovine serum, 2

mM L-glutamine, 100 units/ml penicillin, and 100.g/ml streptomy-

cm. Peritoneal cells were harvested by sterile peritoneal lavage

using H anks’ balanced salt solution, washed, resuspended in culture

medium, and plated at 5 i05 cells/ml. N onadherent cells were

removed by repeated washing after a 2-hr incubation at 37#{ 176}

N it r i t e q uan t i f i cat i on . N 02 accumulation was used as an in-

dicator ofNO production in the medium as described previously

Peritoneal macrophages andRA W 264.7 cells were plated at 5 x i

cells/ml onto 24-well culture plates and stimulated w ith LPS (200

ng/ml) in the presence or absence of #{ 176} -THC (2.5, 5, 10, and 20xM )

for 24 hr. T he isolated supernatants were mixed w ith an

volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene

diamine dihydrochloride, 2% phosphoric acid) and incubated at

temperature for 10 mm. U sing N aNO2 to generate a standard c

nitrite production was measured by an absorbance reading

nm.

Pr epar at i on of I S for R T -PC R . A nrtificial/recombinant

mRNA (rcRNA ) was used as an I S containing specific PCR

sequences for iNOS that were added to RNA samples in a di

series. A rat j3-globin sequence was used as the spacer gene

CB1, CB2, and iN OS IS. This method, developed by V anden H eu

avoids sample-to-sample variation in reference gene expression (e

3-actin) as well as gene-to-gene differences in amplification eff i-

ciency (20, 21) . T he primer sequences from 5’ to 3’ for CB1 and

are forward primer, ACCTGATGTTCTGGA TCGGA ; reverse primer,

TGTFA TCTAGAGGCTGCGCA ; and forward primer, T TCTTACCT -

GCCGCTCATG; reverse primer, CGGATCTCTCCACTCCGTAG, re-

spectively. T he primer sequences from 5’ to 3’ for iN OS are forw

primer, GGATAGGCAGAGA TTGGAGG; and reverse primer, AA T -

GAGGA TGCAAGGCTGG. The CB1 and CB2 IS primer design fro

5’ to 3’ is as follows: IS forward primer, T 7 promoter (TA AT

GACTCACTA TAGG); CB1 or CB2 forward primer, as stated ab

and rat f3-globin forward primer, CCTGCAGTGTCTGA TATTGTTG;

and IS reverse primer, poly d(T )18; CB1 or CB2 reverse primer,

stated above; and rat f3-globin reverse primer,CACACCATTGC-

GA TGAA . The iNOS IS primer design from 5’ to 3’ is as follows:

forward primer, T 7 promoter; iN OS forward primer, as stated

and rat f3-globin forward primer, A AGCCTGGATACCAACCTGCC;

and IS reverse primer, poly d(T )18; iN OS reverse primer, as s

above; and rat globin reverse primer, A ACCTGGA TACCAACCT -

GCC. PCR reaction conditions for generating the I S were perform

as stated previously using 100 ngf rat genomic D NA (21). PCR-

amplified products were purified using the PCR Prep D NA pur

tion system (Promega) and transcribed into RNA using the Gemin

i n vi tr o transcription system (Promega). The rcRNA was subs

quently treated w ith RNase-free D Nase to remove the D NA

plate. A fter quantification, the I S concentration was calculated;

330 x bp is an approximation for the molecular weight ofthe IS

the concentration is expressed in molecules of IS/M I .

Q uan t i t at iv e RT -PCR . RNA was isolated using T n Reagent

(M olecular Research Center, Cincinnati, OH ) as described by C

czynski and M ackey (22). T o avoid any D NA contamination, RNA

samples were incubated w ith RNase-free DN ase for 15m at 37#{ 176}n

10 mM M gCl2, 1 mM dithiothreitol, 25 units of RNasin, 10 mM Tris

and 1 mM EDTA . RNA was then extracted by phenol/chloroform and

precipitated. Competitive RT -PCR was performed according to

land et al . (23, 24) , except that rcRNA was used as an IS instead

genomic DNA . Briefly, total RN A (100 ng) and IS rcRNA of

amount were reverse-transcribed into cDNA using oligo(dT )15 as

primer. Eight aliquots of RNA were taken from each treatm

groups, and an aliquot of IS rcRNA ranging from iO to 10’mole-

cules was added to the appropriate treatment group RNA aliquo

PCR mix consisting of PCR buffer (20 mM ammonium sulfate, 6

T ris, 6.7 M M EDTA , 80 g/ml BSA , and5 mM mercaptoethanol) , 4

m M gCl2, 6 pmol each ofiN OS forward and reverse primers, and

units of Taq DNA polymerase was added to the cD NA sampl

Samples were then heated to 94#{ 176}or 4m and cycled 30 times at

94#{176}enaturing step for 15 sec, a 55#{ 176}nnealing step for 30 sec,

72#{176}longation step for 30 sec, after which an additional extens

step at 72#{ 176}or 5mm was included. PCR products were electropho-

resed in 3% N uSieve 3:1 gels (FM C Bioproducts, Rockland, M E

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3 3 6 J e o n e t aL

visualized by ethidium bromide staining. The CB1, CB2, and iN OS

primers produce amplified products at 450, 429, and 379 bp from the

RNA sample and at 371, 370, and 301 bp from the IS rcRNA ,

respectively. Quantif ication was performed using the Gel Doe 1000

(Bio-Rad, H ercules, CA ), w ith which the amountf target mRNA

present is determined according to G illi landet al . (23) . For example,

the ratio ofthe volume ofthe IS rcRNA to iNOS RNA band is plotted

against the amount of IS rcRNA (in molecules) added to each reac-

tion. T he point at which the ratio of I S (rcRNA ) to iNOS mRNA is

equal to 1 is denoted the “cross-over” point and represents the

amount of iN OS mRNA present in the initial RNA sample. A fter

performance of the 103 0I I range-finding experiment, a second set

of much-narrower IS dilutions (i.e., for iNOS iO#{ 176} - iO’ #{ 176}olecules/

tube) were examined to more accurately quantify RN A message

levels.

EM SA . N uclear extracts were prepared as described previously

(25). T reated and untreated RAW 264.7 cells were lysed with hypo-

tonic buffer (10 mM HEPES, i.5 mM M gCl2 pH 7.5), and the nuclei

were pelleted by centrifugation at 3000< g for 5 mm . N uclear lysis

was performed using a hypertonic buffer (30 mM HEPES, 1.5 mM

M gCl2, 450 mM KCI , 0.3 m EDTA , 10% glycerol) that contained

mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml

concentration each of aprotinin and leupeptin. A fter lysis, the sam-

pies were centrifuged at 14,500 xfor 15 mm , and the supernatant

was retained for use in the D NA binding assay. Two double-stranded

deoxyoligonucleotides containing the CRE (5’ -TGACGTCA -3’ ) (26)

and the NF-K B binding site (5’ -GGGGACTTTCC-3’ (27) were end-

labeled with [y-32P]d.A TP. N uclear extracts (3 g) were incubated

with poly(dI -dC ) (0.5 g for CREB /ATF binding and 2g for NF-K B/

Rel binding) and the 32P-labeled D NA proben binding buffer (100

mM NaC1, 30 mM HEPES, 1.5 mM M gC12, 0.3 m ED TA , 10% glyc-

erol, 1 mM dithiothreitol, 1 mr phenylmethylsulfonyl fluoride, 1

jxg/ml concentration each of aprotinin and leupeptin) for 10m on

ice. D N A binding activity was separated from free probe using a 4.8%

polyacrylamide gel in ix Tris/Borate/EDTA buffer (89 mM T ris, 89

mM boric acid, 2 mM ED TA ). A fter electrophoresis, the gel was dried

and subjected to autoradiography.

cA M P det er min at i on s. RA W 264.7 cells were washed w ith

RPM I 1640, resuspended in RPM I 1640 with 1 mg/ml fatty acid-poor

BSA (CalB iochem), and adjusted to 5 x iO cells/ml. One-milliliter

aliquots of cells were treated w ith either vehicle (1% ethanol) or

#{ 176} -TH C(2.5, 5, 10, and 20 M M ) and then incubated for 10 mm at room

temperature. T he appropriate cell preparations were then stimu-

lated with 50 M M forskolin for iS mm at 37#{ 176}nC% CO2. A fter

stimulation, the adenylate cyclase was inactivated by the addition of

the cAM P extraction buffer (95% ethanol, 0.01HC I ). S up er natants

were collected and dried. A liquots of reconstituted cell lysates were

quantified for cAM P using a cAM P assay kit (D iagnostic Products,

L os A ngeles, CA ) (28).

Stat ist i cal an al ysis. M ean t standard deviation values was

determined for each treatment group in a given experiment. W hen

significant differences occurred, treatment groups were compared

with the vehicle controls using a Dunnett’ s two-tailed test (29).

R e s u l t s

Cannab in oid r ecep tor exp r essi on . E xp r ession of

mRNA for the cannabinoid receptors CB1 and CB2 was

quantif ied using RT -PCR. Total RN A ofRAW 264.7 cells was

prepared, reverse-transcribed into eD NA , and amplified by

PCR using primers specific for the eDNA of interest. I nAW

264.7 cells, CB2 was highly expressed at -1 x iO molecules/

100 ng RNA , whereas CB1 expression was undetectable.

E f f ect of 9-T H C on f or sk ol i n -st im u lat ed adeny lat e

cy cl ase act iv i t y inRA W 264.7 cel l s. T h e ef f ect of L \#{ 176} -T H C

on forskolin-stimulated adenylate cyclase activity was mea-

sured to indirectly verify whether RAW64.7 cells express

functional cannabinoid receptors (Fig. 1). Forskolin

ment alone markedly activated adenylate cyclase as dem

strated by a 10-fold elevation in intracellular cAM P com-

pared with forskolin-unstimulated cells. Pretreatment o

RA W 264.7 cells w ith X #{ 176} -T HC before forskolin stimulation

significantly decreased cAM P formation by 31% , 45% ,

84% at 5, 10, and 20.M respectively. N o effect on cell viabil-

ity was observed in any of the treatment groups and al

exceeded 85% as determined by eosin Y staining (data

shown).

E f f ect s of 9-T H C on n i t r i t e p r odu ct i on and I N O S

gene exp r ession in m acr op hages. L PS (200 ng/ml) alone

increased the production of nitrite 12- fold over basal levels

in penitoneal macrophages and RAW 264.7 cells (Fig.

Basal levels of nitrite in unstimulated peritoneal macro-

phages and RAW 264.7 cells were <1 nmollml (mean

standard deviation, three experiments). On LPS-stimulation,

nitrite synthesis by peritoneal macnophages and RAW

cells increased by 13- and 26-fold, respectively. Furthermore,

this induction in nitrite generation by LPS was inhibited

#{ 176} -TH C. Consistent with these finding, iN OS mRNA was

detectable in unstimulated RAW 264.7 cells. Conversely,

RNA isolated from RAW 264.7 cells treated for 6 hr with

showed the active transcription of the iN OS gene as dem

strated by RT -PCR. Furthermore, z9-TH C (10 and 20 xM )

inhibited LPS-induced iNOS transcription (T able 1). I n this

same series of experiments, it is notable that a very m

increase in iN OS transcript was observed in experiment

the 5 p.M 9-THC-plus-LPS treatment group; however,

believe this was most likely due to biological variability

cause it was not observed in a subsequent experiment.

Because LPS treatment of macnophages activates both

PK A and protein kinase C signaling pathways, RAW

cells were cotreated with PM A or 8-bromo-cAM P in the

ence of LPS and 9-TH C (20M ) for 24 hr to identify which

C/)

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1 0 2 0

F ig . 1 . In h ib it io n o fc A M P p ro d u c tio n b y . 9 -T HC in fo rs ko lin -s tim u -

la ted R A W 264.7 c e lls . R A W 2 6 4 .7 c e lls a t 5 x iO c e lls /m I w

in c u b a te d w ith e ith e r v e h ic le (0 .1 % e th a n o l) o r . 9 -T H C (2 :5 , 5 , 1 0,

2 0 MM ) fo r 1 0 m m , fo llo w e d b y a 1 5 -m m s tim u la tio n w ith fo rs k o lin

M M ). In tra c e llu la r c A M P c o n c e n tra tio n s a re e x p re s s e d a s th e m e a

s ta n d a rd d e v ia tio n o f trip lic a te d e te rm in a tio n s . *, R e s p o n s e th a t is

s ig n ific a n tly d iffe re n t fro m th e c o n tro l g ro u p a s d e te rm in e d b y

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m e n ts is s h o w n .

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F i g . 2 . In h ib it io n o f n it r it e p r o d u c t io n b y 9 -TH C in LP S

stimulated p e r i t o n e a l c e lls a n d R A W 2 6 4 .7 c e lls . P e rito -

* n e a l a d h e re n t c e lls (A ) a n dR A W 2 6 4 .7 c e lls (B ) w e re

tre a te d w ith 9 -T H G (2 .5 , 5 , 1 0 , a n d 2 0M) in th e p re s -e n c e o f LP S (2 0 0 n g /m l) fo r 2 4 h r. T h e s u p e rn a ta n ts w e

s u b s e q u e n tly is o la te d a n d a n a ly z e d fo r n itrite .a r s ,

m e a n ± s ta n d a rd d e via tio n o f trip lic a te d e te rm in a tio n s . *,

R e s p o n s e th a t i s si gni f i cantl y di f f erent fro m th e c o n tro l

g r o u p as d et erm i ned b y D u n n e tt’s tw o-ta ile d t te s t a t p <

0 .0 5 . O n e o f tw o r e p r e s e n t a t i v e e x p e rim e n ts is s h o w n .

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LP S (2 0 0 n g /m l)

T A B LE 1

In h ib it io n o f iN O 5 g e n e e x p r e s s io n b y 9 - THC in L P S -s t im u l a te d

R A W 264.7 c e l l sRA W 264.7 c e lls w ere tre ate d w ith 9 -T H C (2 .5 , 5 , 1 0 , a n d 2 0 M M )n th e p re s e nc e

o f LP S (2 0 0 n g /m l) fo r 6 h r. T o ta l A N A w a s is o la te d a n d th e m o le c u le s o f IN O S

m R N A w e re q u a n tita te d a s o u tlin e d in M ate ria ls a nd M eth o ds .

i NO S

TreatmentExperiment 1 E x p e rim e n t 2

m o le cu le s x flY /iO U ng R NA

NA ND. ND.

L PS 5.89 10.4

L PS + z9-THG (2.5 M M ) 6 .0 5 6 .4 1

LP S + 9 -T H G (5 M M ) 6 .6 2 4 .7 3

LP S + 9 -THG (1 0 M M ) 3 .8 2 4 .2 9

LP S + 9 -T H G (2 0 M M ) 3 .6 5 2 .2 7

N D. , n o t d ete cta b le .

of the tw o potenti al si gnal i ng pathw ay s may be invol ved in

the inhibi t i on ofni tr i c ox i de production by cannabinoids (Fig.

3) . 8-bromo-cA M P, a membrane-permeable cA M P analog, but

not PM A restored ni tr i te production i n the presence of

THC.

Inhibition ofNF-.cB/Rel binding activity by {176}-T H C

treatm ent in L PS-stim ulated RA W 264.7 cells. It has

been reported that protei n binding at the K B binding si te i s

necessary to confer i nducibi l i ty by L PS of iN OS (12). I n the

present ser ies of experiments, w e investigated the rol e of

adenylate cyclase inhibi ti on by 9-TH C on regulati on of NF-

KBIRe1 and the rel ationship betw een NF-K B IRe1 and L PS-

i nduced iNOS gene expression. Our i ni ti al studies demon-

strated that L PS (200 ng/m l) treatment of RA W 264.7 cel l si nduced a marked increase in N F-K B IRe1 binding to i ts cog-

nate si te at 1 and 2 hr, w hich could be v i sual i zed as tw o

distinct bands (Fig. 4). T his banding pattern w as sim i l ar to

that prev i ousl y descri bed by X iet al . (12). I n thei r study , the

upper band w as composed of p5OIRelA (p65) and p50/c-rel

heterodimers, w hereas the low er band consi sted of a p50

homodimer as identi f i ed by gel supershi f t assays (12). I n the

presence of A 9-TH C, L PS-i nduced NF-K B IRe1 binding w as

noti ceabl y i nhibi ted at 1 and 2 hr, and thi s i nhi bi t i on by

-T H C w as dose related, as show n in Fig. 4B . T o confirm the

i nvol vement of N F-K B IReI i n the inducti on of iN OS gene

expression by L PS-stimulated RAW 264.7 cel l s, w e used

F i g . 3 . R e v e r s a l o f 9 -TH C -m o d ia t o d in h ib it io n o f n it r it e p r o d u c t io n

L P S - s t i m u l a te d RA W 264.7 cel ls by 8-bromo-cA MP. R A W 2 6 4 .7 c e lls

w e re tre a te d w ith P M A o r 8 -b ro m o -c A M P in th e p re s e n c e o f LP S

n g /m l) a n d 9 -T H G (2 0 M M) fo r 2 4 h r. T h e s u p e rn a ta n ts w e re s u b s e -

quently isolated a n d a n a ly z e d fo r n itrite . Ba r s , mean ± standard d e v i -

a tio n, trip lic ate d e te rm in atio ns ; *, Significan t d iffe re n c e fro m th e c o n tro l

group as d et erm in ed b y D u n n e tt’s tw o- ta ile d t te s t a t p < 0.05. O n e o f

tw o re p re s e n ta tiv e e x p e rim e n ts is s h o w n .

PDTC, an anti ox i dant that i nhi bi ts N F-K B /Rel acti vati on

( 3 0 ) . Concomitant treatment of RA W 264.7 cel l s w i th PDT

and L PS signi f icantl y inhi bi ted NF-K B IRe1 binding acti v

(Fi g. 5A ). U nder i dentical condi ti ons (i .e., prei ncubati on

PDTC), L PS-activ ated RAW 264.7 cel l s ex hibi ted a dose

dependent i nhi bi t i on in ni tr i te generation, conf i rm inginvol vement of N F-K B /Rel i n L PS-i nduced ni tr i te generati on

(Fig. 5B ). Col l ecti vel y , thi s seri es of ex periments i ndi ca

that N F-K B IRe1 is posi ti vel y regulated by the cA M P casc

to help i ni t i ate iN OS gene expression in response to L P

A ctivation ofCREB/ATF binding w ith L PS in RA W

264.7 cells. In addition to the regulation of N F- fR el,

cA M P signal i ng cascade i s best k now n for i ts acti vati on

CREB/ATF f am i l y ofDN A binding protei ns. B ecause 9 -THC

inhib i ts cA M P signal i ng through the inhibi t i on of adeny lat

cycl ase, the present seri es of experiments w ere perf ormed

inv esti gate w hether CREB /A TF DNA binding factors

acti vated by L PS stimulati on of macrophages and, i f so,

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A )

THC (liM)

5 10 20

LPS- + + + +

B)

FreeproDe)

r eeprone>.

33 8 J eo n e t a L

T im e (mm) 0

LP S -

TH C -

60 120

+ + + +

- + - +

F i g . 4 . In h ib i t io n o f N F-K B /R e l b i n d in g b y z 9 -THG

LPS-st imulated RAW 264 .7 cells. RA W 264 .7 ce lls were

t r eated w it h (A) 9 -THG (20 MM ) in the pre sence of LPS

(200 ng/m l) for 1 or 2 hr or (B ) 9 -THG (2 .5 , 5 , 1 0 , and

MM ) for 2 hr. N ucle ar e xtra cts were then prepa redsubje cted to EM SA. A r r o w h e a d s , N F-KB/R el binding.

R esults a re representa tive of thre e sepa ra te exp

ments.

w hether th eir regu lation w ould a lso be adversely a ffec ted b y

#{176 }-TH C . N uclear ex trac ts w ere prep ared from RAW 264 .7

ce lls stim ula ted w ith LPS (200 ng /m l) fo r various len g ths o f

tim e (0 -2 .5 hr) an d then sub jec ted to EM SA using a CRE

doub le -s tranded o ligon uc leo tide , the co gna te b ind in g s ite fo r

th e CREB /A TF fam ily o f nuc lear facto rs. RAW 264 .7 ce lls

w ere fou nd to ex press a m odera te constitu tive leve l o f CRE

bind ing ac tiv ity befo re LPS s tim u la tion tha t su bstan tia lly

in creased 1 hr a fte r LPS trea tm en t and peaked a t 1 .5 hr . B y

2 .5 hr af te r LPS s tim ula tion , CR E bind ing decreased to basa l

leve ls (F ig . G A ). C RE bind in g w as v isu alized as tw o b ands: a

m ajo r bo ttom band and a m inor top band th at w as o n ly

ap paren t in LPS-ac tiva ted RAW 264 .7 ce lls. In the p resence

of #{17 6}-TH C (20 p .M) , LPS-in duced CRE bind ing w as d im in-

ished a t 1 and 2 h r w ith a lm o st com ple te abroga tion of the

m in or band (F ig . G B ). T he sp ecific ity o f the re ta rded band sw as co nfirm ed by the add ition of an excess o f 32P -un labe led

doub le -s tranded CRE oligonu cleo tide tha t com peted fo r p ro -

te in b ind ing (F ig . 6C ).

E f f ects of #{ 176} -T H C on NF-icBfRel and CREB /A T F bind-

ing act i v i t y i n for sk ol i n -st im u lated RAW 264 .7 cel ls. T o

fu rthe r charac te rize the ro le o f the cA M P signa ling cascade

in the m odulation of nu clea r fac to r b ind in g toB and CRE

m otifs, R AW 264 .7 ce lls w ere treated w ith fo rsk o lin , an agen t

tha t e leva tes cA M P by direc tly ac tiva ting adeny la te cy clase.

A s show n in F ig . 7 , bo th N F -K B IR e1 an d CR EB /A TF bind ings

w ere enhanced a t 30 and 60 m m afte r fo rsk o lin trea tm en t.

H ow ever, stim ula tio n of ce lls w ith fo rsko lin in th e presence

of #{176} -TH C (20 M ) resu lted in th e inh ib itio n ofnuc lea r fac to r

b ind ing to bo th KB and C RE .

D i s c u s s i o n

W e dem ons tra ted th at 9 -TH C treatm en t s ign ifican tly a t-

ten ua tes LPS -indu ced iN O S transc rip tion th rough an inh i-

b itio n of the cA M P signa lin g cascade in the m acrophag e line

RAW 264 .7 . A s prev ious ly show n by a num ber of resea rchers,

in clud ing o urse lves , lym pho id an d m yelo id ce lls exp ress can-

nab ino id recep to rs (3 , 4 , 7 , 8 , 3 1), w h ich nega tive ly reg u la te

ad eny la te cyc lase and lead to a decrease in ce llu la r cA M P (6 ,

32 , 33 ). L igand b in d ing to these recep to rs b y can nab ino ids

rep resen ts o ne of th e pu ta tive m echan ism s by w hich th is

c lass o f com pounds exerts the ir b road range o f b io log

effec ts , inc lud ing imm une sup press ion . A lthough the re la

d is tribu tion of th is fam ily of recep to rs on sp ec ific ce ll

w ith in th e imm une sys tem has no t been w ide ly stu

rad io lig an d b in d ing an a ly sis ind ica tes tha t the re a re

recep to r b ind ing site s in m ou se sp leen ce ll p repa ratio ns

P rog ress h as been m ad e in desc rib ing w hich of the tw o

n ab ino id recep to r typ es , CB 1 and /o r CB 2 , is expres sed w i

the imm une system and o ther tis su es. R esu lts o f rad io ligan

b in d ing , N o rth e rn ana lysis , and s truc tu re -activ ity re la tion -

sh ip s tud ies s trong ly sug gest th a t CB2 is the pred om inan t

fo rm of cannab ino id recep to r expressed w ith in the imm

system (3 , 4 , 7 , 31 ). It is n o tab le tha t CB1 , a lthou gh

iting low expression a t the transc rip tio na l leve l, h as been

iden tified in a v ar ie ty o f imm unolog ica l p rep arations by RT -

PCR (3 , 7). In these experim en ts, exam ina tion o f RAW 2

cell R NA by qu an tita tiv e RT -PCR revea led tran sc rip ts

CB 2 on ly an d no de tec tab le CB 1. T he fun c tion ality o f ca

b in o id recep to rs in RAW 264 .7 cells w as verified

ab ility o f z 9 -TH C to inh ib it fo rsk o lin -stim ula ted cAM P accu-

m ula tion in a dose-re la ted m anner.

R ecen tly , it w as dem ons tra ted th a t in itiation of iN O S

scrip tio n is un der the con tro l o f the cAM P cascade .

m en t o f various ce ll p repara tio ns, inc lud in g ra t pe ritonea l

m acrop hages (18) and vascu la r sm oo th m u sc le ce lls

w ith m em brane-perm eab le cA M P ana log s (d ibu ty ry l-cA M P

and 8-brom o-cA M P ) or w ith fo rsko lin resu lted in an augm

ta tion in N O syn thesis . M ost p er tinen t to our stud ies ,

b een show n in m acrop hages tha t induc tion of N O by ib le com plexes of m onoc lo na l IgE an ti-d in itropheno l an tibo dy

and d in itroph en o l-BSA is m ed ia ted th rough an increase

iN O S exp ress io n tha t is in dependen t o f eleva tions in

cy toso lic calc ium or pro te in ty ros in e p hosphory la tion (1 8).

C ons is ten t w ith these find ing s, our experim en ts show

9 -THC , a t co ncen tra tion s tha t inh ib it ad en y la te cyc lase ,

s im ultaneo usly inh ib its LPS-ind uced N O pro duc tion in

RAW 264 .7 ce lls and perito neal m acrophag es . F urthe rm o re,

th is cann ab ino id -m ed ia ted in h ib ition of N O p roduc tion

b e abro ga ted by concom itan t trea tm en t o f RAW 264 .7

w ith 8-b rom o-cA M P but no t w ith pho rbo l este r. T hese

ings sug gest the invo lvem ent o f the cAM P cascade

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C/ )

0)

0

(0

0

*

0)

0

EC

: *

z

T im e (m m ) 0

LP S -

TH C -

6 0 1 2 0

B)

C )

+ + + +

- + - +

. #{149}.

9 -THC In h ib i t s L P 5 -ln d u c ed iNOS an dN F -,c B /R e l B in d in g 33 9

A )

B )

F r e eprobe)

LP S - + - +

A )

Free

probe>.

Tim e (h) 0 0 . 5 1 1 . 5 2 2 . 5

N A 0 5 10 25 50

P D TC M)

LP S (2 0 0 ng/ml)

F ig . 5 . In h i b i t io n of N F -KB /R e l b in d in g a n d n itrite p ro d u c tio n b y P D TC

in LPS -stim ulated R A W 264.7 cells. A ,R A W 264.7 cells w ere treated

w ith PD T C (5 0 MM ) in th e p re s e n c e o r a b s e n c e o f LP S (2 0 0 n g /m l) fo r 2

hr. N uclear extracts w ere then prepared and subjected to EM S A .Ar-

r o w h e a d s , N F -KB /R e l b in d in g . B ,R AW 2 6 4 .7 c e lls w e re tre a te d w ith

PD TC (5, 1 0, 25, and 50 M) in th e p re s e n c e o f LP S (2 0 0 n g /m l) fo r 2 4

h r. T h e s u p e rn a ta n ts w e re s u b s e q u e n tly is o la te d a n d a n a lyz e d fo r

nitrite. Each bar represents the m ean ±tandard deviation for triplicate

determinations. *, Significant difference from the control group as

determ ined by D unn ett’s two-tailed t te s t a t p < 0 .0 5 . R e s u lts a re

r e p r e s e n t a t i v e of three s e p ara te e xp er im e n ts .

regulation of iN O S by A W 264.7 cells as previously sug-

gested by studies in rat peritoneal m acrophages (18).

T he activation ofN O pr oduct ion is closely t ied to tr anscr ip-

tional activation of iN O S. Insight into the regulation of the

iN O S gene com es fr om the r esults of r ecent studies that have

identified several know n nuclear regulatory factor recogni-

tion m otifs w ithin the iN O S prom oter region. D eletion anal-

ysis of the iN O S prom oter using luciferase reporter con-

structs identified tw o regions that significantly increased

lucifer ase activity inR A W 264.7 cells after LPS treatm ent

(term ed regions I and I I ) (11) . R egion I (position -48 to

- 209 ) contains binding m otifs for N F-K B and N F-interleu-

kin-6 and was found to be essential for lucifer ase activity.

R egion II (position -913 to 1029) contains m otifs for bind-

L PS - + +

coKB - - +

’1 :

)..

F ig . 6 . 9 - THG -med i a t e d inhibition o f C R E B /ATF b in d in g in LP S -s tim -

ulated R AW 2 6 4 .7 c e lls . A , N u c le a r e x tra c ts fro m LP S (2 0 0 n g /

stim ulated R A W 2 6 4 .7 c e lls w e re in c u b a te d w ith 3 2 P -la b e le d G

p r o b e . B , R AW 2 6 4 . 7 c e l l s w e re tre a te d w ith z 9 -THG (2 0MM ) in th e

p r e s e n c e of LP S . C , In c om pe titio n studies, 1 pm ol of n la b e le d KB o r

G R E w a s a d d e d to th e re a c tio n m ix tu re . R e a c tio n p ro d u c ts w e re e

trophoresed, a n d th e g e ls w e re d rie d a n d a u to ra d io g ra p h e d . Arrow-

h e a d s , CREB /ATF b in d in g . R e s u lts a re re p re s e n ta tive o f th re e s e p a ra te

experim ents.

ing IFN -y-related transcription factors (IFN -stim ulated re-

sponse elem ent and Pu.1JIFN --y elem ent) as w ell asB

m otif. C onstructs containing region II alone exhibited

activity in L PS -activated R A W 264.7 cells. C onstructs con-

tam ing both regions I and II exhibited a greater m agnitude

activity than found in region I alone, and activity w as fur

enhanced by cotreatm ent w ith IFN --y. These studies sugg

that region II functions as an enhancer for region

involvem ent of N F-K B IR e1 fam ily m em ber proteins in

gene regulation by m acrophages in response to LPS-stim u-

lation w as confirm ed by gel shift studies and iN O S luciferas

constructs using R A W 264.7 cells (12). D eletion of K B m

from iN O S constructs resulted in the abrogation of luciferas

activity after LPS treatm ent (12). G el shift and supershi

studies dem onstrated that LPS treatm ent induced a rapi

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30 60

+ + + +

Tim e (m m ) 0

F S K -

A)

B )

Refe rence s

1 . H erk en ham , M ., A . B . Lynn , M . D . L ittle , M . R . Joh nso n , L . S . M elv in

R . de C os ta , a nd K . C . R ic e. C annabin o id rec ep tor loc aliz atio n in

Pr oc. Nati . Acad. Sci . U SA87 :19 32-1936 (1 990).

2 . T hom as , B . F ., X . W e i, an d B . R . M ar tin . C ha rac ter izat ion an d au to rad

g rap h ic loc aliz atio n o f the can nab ino id b ind ing s ite in ra t b rain

[ 3H ]1 1 -OH -19 -THC -DMH . J. Pharmacol . Exp. Ther . 263:1383-1390

(1992) .

3 . K am insk i, N . E ., M . E . A bood , F . K . K e ssle r, B . R . M art in , an d

Schatz . Iden tif ica tion of a func tio nal ly relevan t cannabin o id rec ep tor

m ouse sp le en ce lls th at is in vo lved in c ann ab ino id-m ed iated im m une m od-

u lat ion . M ol . P har macol . 42:7 36-742 (1992).

4. M unro , S ., K . L . Thom as , an d M . A bu -Sh aar . M o lecu lar ch ara cter iza tion

a pe riph era l recep to r for can nab ino ids . Natur e (L ond.) 365:6 1-6 5 (19 93) .

5. M atsu da , L . A ., S . J. L o lai t, M . J. B row n ste in , A . C . Y oung , and

Bonn er. S tru ctu re of a cannabin o id rec ep tor and fun ctiona l exp res sio n

the c lo ned eDN A . Natur e (Lond.) 346 :561 -564 (199 0 ).

6 . K am insk i, N . E ., W . S . K oh , M . L ee, K . H . Y ang , and F . K . K

Supp ressio n of the h um ora l im m un e respon se by cann abin oids is pa r

media ted thro ug h in h ib itio n of adenylate cyclase b y a pertuss is

sens itive G -pro te in co upled m echanism . Biochem. Pharmacol . 48:1899-

1 9 0 8 ( 19 9 4 ) .

34 0 J e o n e t a L

F i g . 7. Inhibition of N F-K B /R el and G R E B /A TF binding by -TH G in

fo rsko l in -s t imula ted R A W 264.7 cells. R A W 264.7 cells w ere treated

w ith -TH G (20 M M ) in the presence of forskolin (50M ) for 30 or 60

mm . N uclear extracts w ere then prepared and incubated w ith 32P -

labeled (A ) K B probe or (B ) G R E probe. R eaction products w ere elec-trop horesed , and th e ge ls w ere dr ied an d au to rad io graphed . Arrow-

h e a d s , C R E B /A TF and N F-K B/R el binding. R esults are representative of

th ree separa te experim en ts.

(30 mm ) and persis ten t (m easurab le a t 2 hr) ac tiv a tion of

sev eral N F-K B IR e1 fam ily m em ber com plexes, inc lud ing he t-

e rod im ers o f p50 /c -re l, p5O fR el A (p65 ), and p 5O /p 5O ho-

m odim ers (12). A s d iscussed , o ur experim en ts show tha t

9 -THC effec tive ly inh ib its LPS ac tiva tion of iN O S transc rip -

tion and N O activ ity in RAW 264 .7 cells . T o fu rthe r investi-

ga te the pu ta tive m echan ism by w h ich can nab ino ids inh ib it

iN O S , w e cho se to m on ito r the effec ts o f cannab ino id com -

pounds o n the activa tion of tw o fam ilies o f transc rip tion

fac to rs b y ge l sh ift a ssays d uring LPS ac tiva tion of m acro -ph ages. N F -K B IR e1 fam ily m em ber b ind ing ac tiv ity w as ex-

am ined in lig h t o fthe ir cr itica l ro le in the regu la tio n of iN O S .

A lso , C REB /A TF bind in g w as m onito red as a m easure of

inh ib ition of the cA M P signa ling cascade b y can nab ino ids .

K ine tics stud ies show ed s trong ind uc tio n b y LPS of tw o sep-

a ra te KB b in d ing com p lexes a t 60m and m o re enh anced

indu c tion a t 120 mm . z #{176} -THC inh ib ited the ac tiv ation o f bo th

of these K B b in d ing com plexes; how ever, the m agn itud e of

inh ib ition seem ed to be grea te r fo r the pro te in com plex rep -

resen ted by the top o f the tw o band s. T h is find ing sugges ts

tha t 9 -TH C m ay inh ib it the fo rm ation of e ithe r p5 0 /c- re l o r

p5O fR el A (p 65) he te rod im ers based on the study by X iet al .

(1 2) in w hich the au tho rs id en tified these N F -K B fR el p ro teins

as be ing resp ons ib le fo r th is top band . O ur experim en ts ver-ified tha t the inh ib ition of LPS -induced KB b ind in g by

THC w as dose dependen t and th a t the b ind ing w asB spe-

c ific as d em onstra ted by com petition assays w ith 32P -

un labe led KB p robe . A dd itiona l co n tro l exp erim en ts w ere

perfo rm ed to verify a ro le fo r N F-K B /R el fam ily m em bers in

iN O S reg u la tion us ing th e PD TC , an inh ib ito r o f N F-K B

ac tiva tion , in our LPS-trea ted RAW 264 .7 ce ll p rep arations

(3 0). PD TC effec tive ly inh ib ited the ac tiva tion of N F -K B IR e1

b in d ing com plexes and N O produc tion .

B ecau se ofthe inh ib ito ry effec ts th at cannab ino id s exert on

the cA M P form ation an d s igna ling coup led w ith the fac t th a t

LPS-in duced N F-K B ac tiva tion h as b een reported to be pri-

m an ly reg u lated by PK A (14 , 15 , 34) , ou r experim en ts

focused on cAM P signa ling . T rea tm en t o f RAW 264 .7

w ith e ith er LPS o r the ad en y late cyc lase ac tiva to r fo rsk o

resu lted in th e ac tiv ation of CR E bind in g pro te ins .

ac tiva tio n s tim u li in duced tw o CRE bind ing com plexes

w ere v isua lized as a m inor (top ) an d a m ajo r (bo ttom ) ban

g e l sh ift a ssays . L ikew ise, ac tiva tio n ofRAW 264 .7 ce lls

e ithe r s tim uli (i.e., L PS or fo rsko lin ) in the presence

THC resu lted in a decrease in C RE bind ing of bo th o

D N A bind ing com p lexes and is ind ica tive th at 9 -TH C

duces an inh ib ition in the cAM P signa ling cascade . F urth

m o re , concord an t w ith prev iou s stud ies sug gesting tha tKA

is respons ib le fo r the ac tiva tion of N F -K B IR e1 as show n

m acro phages (1 4 , 15) and B ce lls (34), trea tm en t o fAW

264 .7 ce lls w ith fo rsko lin resu lted in the ac tiva tion of N

b in d ing tha t w as also a ttenua ted by 9 -TH C . A dd ition

ev id en ce tha t cAM P signa lin g is inv o lved in LPS -indu ced

respon ses by m acroph ag es w as pro v ided by th e ac tiva tion

CRE b ind ing afte r LPS trea tm en t o f RAW 264 .7 ce lls .

L PS-ind uced ac tiv a tion ofCR E D N A bind in g pro te ins pea

a t --90-1 20 mm afte r LPS trea tm en t and resu lted

s im ila r band in g pa tte rn to tha t o bse rv ed afte r fo rsko

s tim ula tion . T ak en toge ther, ou r f in d ings s trong ly sug

tha t N F-K B fR el is ac tiva ted by the cA M P signa ling cas

b y PK A -m edia ted pho spho ry la tio n of the N F -K B inh ib ito ry

pro te in , 1KB ( 35 ) .

In summ ary , these ex perim en ts dem ons tra te tha t 9 -T

inh ib its LPS -induced ac tiva tion of iN O S in m acro phages

N O p roduc tion . B ased on our find in gs, the m o st lik e ly

an ism th at can accoun t fo r th is b io lo g ica l e ffec t inv o lves

inh ib ition of cAM P form ation th rou gh nega tiv e reg u lation

adeny la te cyc lase by G pro te in -coup led cannab ino id recep-

to rs. Inh ib ition of the cAM P sign aling cascad e a ttenu ates

ac tiv ation o fN F -K B b ind ing pro te ins, w h ich are necessa ry

the activa tion of the iN O S gene . A t leas t tw o s ign ifica

po in ts a re b ro ugh t ou t by th ese s tu d ies. F irst, these exm ents fu rthe r confirm the cr itica l ro le o f cA M P sig na ling

the reg u la tion of iN O S v ia N F -K B . F urthe rm o re , du e to the

critica l ro le tha t N O release p lays in m ed ia ting in flamm ato ry

responses, the inh ib ito ry effects o f cann ab in o ids o n iN O S

sugges t th at th is fam ily ofcom pounds m ay represen t a u

new class o f an ti-in flamm atory ag en ts . T h is is fu rthe r sup

po rted by ou r recen t find in gs tha t cannab in o id com pound

a lso inh ib it in te rleuk in -2 , a c ritica l m ed ia to r o f T ce ll c lona l

expans ion (36).

8/2/2019 Inos Attenutation by Delta

http://slidepdf.com/reader/full/inos-attenutation-by-delta 8/8

9 -TH C In h ib it s LP 5 -In d u c e d iN O S a n d N F -ic B /R e I B in d in g 341

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Send r epr int r equests to:Norbert E. K aminsk i , Ph.D ., D epartment of Ph

macology and T ox i col ogy , M ichigan State U ni v ersi ty , B 330, L i f e ScB ui ld ing, East L ansing, M I 48824. E-mai l : k am i nsi 1t4pi lot .msu.edu